This invention is directed to peptides with various physiological activities, particularly wound healing.
Wound healing is a complex biological process that differs according to the wound type: acute or chronic. The principal elements of wound repair are the immediate events of hemostatis and stimulus for inflammation, then inflammation and cell proliferation and migration, then followed by molecular synthesis, collagen polymerization and cross-linking, remodeling, and wound contraction. Inflammation is characterized by vasodilation, increased vascular permeability, leukocyte infiltration, bacterial killing, and macrophage-based stimulation of cellular proliferation and protein synthesis.
In cell proliferation and migration, fibroblasts appear within 2-3 days and dominate wound cell population during the first week. For the initial 2-3 days, their activity is confined to fibroblast replication and migration. At days 4-5, fibroblasts begin to synthesize and secrete extracellular collagen. Fibroblasts produce GAG and collagen.
Angiogenesis is essential to wound repair and scar formation. Capillary proliferation is required to support fibroblast migration into wound and fibroblast metabolic requirements. In the absence of angiogenesis, such as in ischemic ulcers or arteriosclerosis obliterans, fibroblast migration arrests and wound healing fails to proceed.
Angiogenesis has the stages of cell attachment, basement membrane degradation and migration, proliferation, and differentiation, and is associated with epithelial cell migration.
Molecular synthesis includes collagen synthesis and proteoglycan synthesis. Collagen synthesis begins with the intracellular phase of monomer synthesis. Secretion into the extracellular space then occurs, followed by polymerization into collagen fibers and cross-linking to increase tensile strength.
Remodeling typically begins 3 weeks after injury. Equilibrium between collagen synthesis and degradation is achieved. Wound remodeling begins and will continue for 2 years. There is a progressive increase in tensile strength as Collagen III is replaced by Collagen I. Epithelialization is the hallmark of successful wound repair and occurs in four phases: mobilization, migration, mitosis, and cellular differentiation.
Granulation tissue contains numerous capillaries and has a support matrix rich in fibroblasts, inflammatory cells, endothelial cells, myofibroblasts, and periocytes. If vascular endothelial growth factor (VEGF) is removed, there is an absence of granulation tissue, and wound angiogenesis and the wound healing process cease.
In chronic wound healing, there is typically an absence of epithelial migration, excessive granulation tissue, and fibrosis, with scarring and impaired function possibly being present.
Although many advances have been made in the understanding of wound healing, the healing of wounds still presents a considerable challenge to the clinician. This is particularly true in patients who are diabetic, who have impaired circulation of the skin, or who are susceptible to infection, such as the result of being in an immunocompromised condition. Additionally, when such wounds do heal, they frequently heal with cosmetically undesirable consequences such as scars or discoloration.
Accordingly, there is a need for an improved method of wound healing that is particularly suitable for application in patients with diabetes, who have poor circulation in the skin, or who are immunocompromised. There is a further need for treatments and methods that can reduce or eliminate the consequences that can occur from wound healing, such as scars and discoloration. There is an additional need for factors that are well-tolerated and can be used with other treatments in such patients.
One aspect of the present invention is a substantially purified peptide having a physiological activity, the peptide comprising a sequence from 28 to 44 amino acids, including therein a sequence of L-K-E-K-K (SEQ ID NO: 1), the peptide having a physiological activity selected from the group consisting of wound healing activity, immunostimulant activity, and growth factor activity, wherein the peptide is in linear or cyclic form, and wherein the peptide is in linear form, the amino terminus is optionally acetylated. The sequence within this peptide can be L-K-E-K-K-E (SEQ ID NO:2) or L-K-E-K-K-E-V-V-E (SEQ ID NO: 3).
The invention includes the following peptides:
(1) a substantially purified peptide having wound healing activity, the peptide comprising a sequence of 28 amino acids, that is: Ac-S-D-A-A-V-D-T-S-S-E-I-T-T-K-D-L-K-E-K-K-E-V-V-E-E-A-E-N (SEQ ID NO: 4), where xe2x80x9cAcxe2x80x9d indicates that the amino terminus is acetylated, the peptide being linear;
(2) a substantially purified peptide having wound healing activity, the peptide comprising a sequence of 35 amino acids, that is: K-L-K-K-T-E-T-E-Q-K-N-P-L-E-V-L-K-E-K-K-E-V-V-E-L-K-E-K-K-V-V-I-E-N-P (SEQ ID NO: 5), the peptide being linear;
(3) a substantially purified peptide having immunostimulant activity, the peptide comprising a sequence of 38 amino acids, that is: Ac-A-D-K-P-M-G-E-L-A-S-F-D-K-A-G-L-K-E-K-K-E-T-L-P-T-K-E-T-I-E-E-E-K-R-S-E-I-S (SEQ ID NO: 6), the peptide being linear;
(4) a substantially purified peptide having wound healing activity, the peptide comprising a sequence of 44 amino acids, that is: Ac-A-N-K-G-Q-A-P-G-E-A-M-K-P-Sxe2x80x94F-L-K-E-K-K-E-V-V-E-R-S-K-E-E-E-G-P-A-K-M-N-L-V-I-E-M-P-K-D (SEQ ID NO: 7), the peptide being linear; and
(5) a substantially purified peptide having wound healing activity the peptide comprising a sequence of 44 amino acids, that is: K-K-L-K-K-E-E-N-P-L-E-L-K-E-K-L-K-E-K-K-N-P-L-P-S-K-E-E-E-K-A-S-P-F -D-K-J-T-E-T-P-D-M-S (SEQ ID NO: 8), the peptide being linear.
Another aspect of the present invention is an isolated nucleic acid molecule encoding a peptide of the present invention. Typically, this nucleic acid molecule is DNA.
Yet another aspect of the present invention is a vector comprising DNA according to the present invention operably linked to at least one control element that influences the expression of the DNA
Still another aspect of the present invention is a host cell transfected with a vector according to the present invention such that the cell expresses the peptide encoded by the vector according to the present invention.
Another related aspect of the present invention is a method of producing a substantially purified peptide having a physiological activity comprising the steps of:
(1) culturing a host cell according to the present invention; and
(2) isolating the peptide produced by the host cell to produce the substantially purified peptide.
Yet another aspect of the present invention is a pharmaceutical composition comprising:
(1) a peptide according to the present invention in a physiologically effective quantity; and
(2) a pharmaceutically acceptable carrier.
A particularly preferred pharmaceutically acceptable carrier comprises water, Seamollient, a polymer selected from the group consisting of carboxymethylcellulose and hydroxyethylcellulose, and at least one preservative selected from the group consisting of methylparaben and propylparaben.
Still another aspect of the present invention is a method of use of a peptide according to the present invention for promoting wound healing, stimulating the immune system, or stimulating pituitary/hypothalamus activity in a mammal in need thereof comprising administering an effective quantity of a peptide according to the present invention having the appropriate activity to the mammal.